The present invention relates to crystals of .beta.-nicotinamide-adenine-dinucleotide of free acid type and a process for preparing the crystals.
.beta.-Nicotinamide-adenine-dinucleotide (hereinafter referred to as "NAD") is present as a coenzyme for various oxidoreductases in almost all of the tissues of living bodies, and has a very important role in energy metabolism, biosynthesis, etc. in a living body. Therefore, in recent years, the demand for NAD has increased not only as reagents for research on biochemistry and physiology, but also as chemicals indispensable to clinical diagnosis as a factor of measurement in enzymatic analysis upon measuring enzyme activity and concentration of a substrate.
Hitherto, NAD has been obtained in solid form by isolating NAD from yeast extract or a cultured broth of a microorganism by various methods of the isolation such as ion exchange chromatography and subjecting the obtained solution of NAD to a method such as freeze drying or precipitaion with an organic solvent followed by separation and drying of the precipitate. The thus obtained solid NAD is amorphous, and is very hygroscopic and deliquesces in air. In many cases, such an amorphous NAD still contains a trace amount of impurities. Also, the amorphous NAD is unstable, and lowering of the purity due to thermal decomposition during storage and transportation is unavoidable. It is known that a competitive inhibitor of an enzyme is present in the thermal decomposition fragments and a trace amount of other impurities. Therefore, it is well known that the use of such a NAD of low purity in the enzymatic analysis gives only results having a large error, for instance, from Dalziel, J. Biol. Chem., Vol. 238, 1538(1963).
Crystallization of NAD of free acid type has been reported by A. D. Winer in J. Biol. Chem., Vol. 239, PC3598(1964). However, this process uses a large amount of a solvent and moreover requires a very low temperature, i.e. -15.degree. C. The standard parameters for this process are indefinite and there is no reproducibility. Also, the disclosed crystals are crystals of NAD trihydrate which are long thin needles or flat prisms, and it is reported that the crystalline NAD changes into the amorphous form by the change in surrounding humidity and the stability is bad. Further, the process has the disadvantage that the purified product obtained by the use of a solvent contains a slight amount of the unseparable solvent. Also, the use of a large amount of a solvent is not economical, and the process has no practical importance as an industrial process.
Crystals of a metal salt of NAD such as the lithium salt are also known. However, when NAD free acid is required, the metal salt must be treated again with an ion exchange resin, and accordingly the purification of amorphous NAD by this process is disadvantageous in increase of the process steps.
It is an object of the present invention to provide novel crystals of NAD of free acid type.
A further object of the invention is to provide crystalline NAD having a high purity and a high stability.
A still further object of the invention is to provide amorphous NAD having a high purity.
Another object of the invention is to provide a process for preparing NAD having a high purity in a simple manner in a high yield.
These and other objects of the present invention will become apparent from the description hereinafter.